The present invention is in the field of recombinant DNA technology. This invention relates to vectors which encode stable messenger RNA (mRNA) and methods of using such vectors. In particular, this invention relates to vectors which establish controlled expression of recombinant genes within a tissue; the expression may be at levels which are useful for gene therapy and other applications. The invention further relates to vectors able to express growth hormone releasing hormone (GHRH) and gene sequences inserted into vectors that control the production of growth hormone releasing hormone in non-human vertebrate animals. The invention is directed further to the use of these vectors in the respective non-human animals to further growth and strengthen their immune systems.
None of the information provided herein is admitted to be prior art to the present invention, but is provided only to aid the understanding of the reader.
Growth hormone (GH) secretion by the anterior pituitary is stimulated by growth hormone releasing hormone (GHRH) and inhibited by stomatostatin (SS), both hypothalamic hormones (Scanlong, M. F. et al., 1996, Hormone Research 149-154). GH enhances protein synthesis, lypolysis, and epiphyseal growth, and is implicated in the regulation of the immune system. GH increases circulating insulin-like growth factor I (IGF-1) levels, which in turn, mediates growth in the liver and peripheral tissues.
The GHRH-GH-IGF-I axis undergoes dramatic changes during the aging process and in the elderly (Iranmanesh et al., 1991, J. Clin. Endocrin. and Metab. 73:1081-1088; D""Costa A. P. et al., 1993, J. Reproduction and Fertilityxe2x80x94Suppl. 46:87-98,) with decreased GH production rate and GH half-life, decreased IGF-I response to GH and GHRH stimuli that lead to osteoporosis, increase in fat and decrease in lean body mass and tissue function (Corpas et al., 1993, Endocrine Rev. 14:20-39).
In addition, genetic disorders of growth have also been ascribed to defects in the GHRH-GH-IGF-I axis, as those of GHRH receptor (Cao, Wagner, Hindmarsh, Eble, and Mullis, 1995, Pediatr. Res. 38:962-966), GH gene (Cogan et al., 1993, J. Clin. Endocrin. and Metab. 76:1224-1228; Vnencak-Jones et al., 1988, PNAS 85:5615-5619), GH receptor (Amselem et al., 1993, Human Molec. Gen. 2:355-359; Amselem et al., 1991, Paediatrica Scandinavicaxe2x80x94Supplement 377:81-86; Meacham et al., 1993, J. Clin. Endocrin. and Metab. 77:1379-1383) and pit-1 (Parks et al, 1993, Hormone Research 40:54-61) a pituitary specific transcription factor. In many cases growth retardation (GR) is a secondary manifestation of an unrelated primary affection (Turner syndrome, chronic renal failure, ovary resistant syndrome) or the exact cause of GR can not be established (Parks et al., in Molecular Endocrinology: Basic Concepts and Clinical Correlations (ed. Weintraub, B. D., Raven Press Ltd., New York, 1995) p.473-490).
In these cases of GR where the GHRH-GH-IGF-I axis is unaffected and in elderly, as well as in nonstatural related catabolic conditions (burn, sepsis, trauma associated pathology, chronic obstructive pulmonary disease), GH or GHRH replacement therapy is efficient.
Recombinant GH therapy is currently used in clinics, but a large number of studies have shown that side effects occur frequently, including edema, hypertension, carpal tunnel syndrome, hyperinsulinemia and impaired glucose tolerance (Marcus et al., 1990, J. Clin. Endocrin. and Metab. 70:519-527; Salomon et al., 1989, New Engl. J. Med. 321:1797-1803).
GH and IGF-1 also have beneficial effects on immune function (LeRoith, D. et al., Endocrinology 137:1071-1079 (1996)); Kotzmann, H. et al., Neuroendocrinology 60:618-625 (1994)). In farm animals, GHRH is galactopoietic (stimulates milk production) with no alteration in milk composition, increases the feed to milk conversion and sustains growth, mostly through increased lean body mass (Enright, W. J. et al., Journal of Animal Science 71:2395-2405 (1993); Enright, W. J. et al., Journal of Dairy Science 69:344-351 (1986)).
Studies have shown that relatively small amounts of GHRH are required to stimulate the production and secretion of GH in all species. Some benefits of increasing GH in non-human vertebrate animals are improved growth rates, an increase in lean body mass, an increase in feed efficiency in pigs, beef cattle and sheep, increased milk production in dairy cows and goats, and enhanced production of lean meat and egg production in poultry.
GH also enhances the immune system in animals. In animals GHRH will have a great therapeutic utility in the treatment of cachexia in chronic diseases such as cancer, diabetes, due to growth hormone production abnormalities, enhancement of burn and wound healing, bone healing, retardation of the aging process and osteoporosis. However, the greatest use will be in agricultural animals. Intramuscular injection of DNA vector can persist for several months to produce sustained levels of GHRH. The intramuscular delivery of GHRH vector represents a practical method for improving performance in livestock animals.
Current limitations of recombinant GHRH therapy are the high cost of recombinant proteins, the short half-life of the peptides in vivo and the requirement for frequent administration (1-3 times/day) of either subcutaneous or intravenous injections. Using a GHRH injectable DNA plasmid based vector will enhance endogenous GH secretion in vertebrate animals with GH deficiencies in a manner more closely mimicking the natural process and in a less expensive manner than classical therapies.
The present invention is based in part on the identification of certain nucleic acid sequences which confer advantageous tissue targeting, expression, and secretion properties. Such sequences are utilized in the construction of plasmid vectors encoding GHRH, for delivery and expression of the GHRH coding sequences.
Expression of these vectors can be tissue specific. These vectors are useful in facilitating enhanced expression in tissues as well as in targeting expression with tissue specificity. These vectors can be used to treat diseases by gene therapy by restricting expression of a gene encoded on the vector to targeted tissues. Vectors containing such sequences are able to provide gene delivery and controlled expression of recombinant genes within tissues; such expression can be at levels that are useful for gene therapy and other applications. These vectors can also be used to create transgenic animals for research or livestock improvement.
The ability of the expression vector to provide enhanced product secretion as well as direct expression to specific tissues allows the vector to be used for treating numerous diseases. The above vectors can be used in gene therapy where a vector encoding a therapeutic product is introduced into a tissue so that the tissue will express the therapeutic product. For example, the above vectors may be used for treating muscle atrophy associated with neurological, muscular, or systemic disease or aging by causing tissues to express certain trophic factors.
In addition, the vectors can be used for gene replacement of inherited genetic defects or acquired hormone deficiencies, for vaccination in humans or animals to induce immune responses, or for creating transgenic animals. The transgenic animals can be used as models for studying human diseases, for assessing and exploring novel therapeutic methods, to assess the effects of chemical and physical carcinogens, and to study the effect of various genes and genetic regulatory elements. Furthermore, transgenic animals can be used to develop commercially important livestock species. The above vectors can also be used to transform cells to produce particular proteins and RNA in vitro.
Expression of such vectors having a GHRH encoding sequence in the body of a vertebrate, e.g., a human, can produce both direct and indirect effects. The GHRH produces direct effects by the direct action of the GHRH polypeptide. However, indirect effects may also be produced due to the effect of the GHRH inducing or turning on the expression of other genes, or modulating the activity of other gene products. In particular, expression of GHRH can affect the levels of GH and IGF-I.
In a first aspect, the present invention features a vector for expression of a nucleic acid sequence in tissue by encoding stable mRNA. The vector includes a 5xe2x80x2 flanking region which includes necessary sequences for the expression of a nucleic acid cassette, which include a promoter sequence, preferably an actin gene promoter sequence, more preferably a skeletal actin gene. The vector also includes a 3xe2x80x2 flanking region, which includes a 3xe2x80x2UTR and/or a 3xe2x80x2NCR, which enhances secretion of the product expressed from the nucleic acid cassette. Preferably the 3xe2x80x2UTR is from the 3xe2x80x2 region of a growth hormone gene, more preferably from a human growth hormone gene. Alternatively, in related vectors, the 3xe2x80x2 sequences may be selected to provide a higher level of retention of the product within a tissue, e.g., within a muscle tissue, rather than enhancing secretion. Such sequences can, for example, be from a skeletal xcex1-actin gene. The vector also includes a linker which connects the 5xe2x80x2 flanking region to a nucleic acid. The linker does not contain the coding sequence of a gene that the linker is naturally associated with. That is, the linker is not the normal gene associated with the 5xe2x80x2 and 3xe2x80x2 regions. Preferably, the linker includes a sequence coding for a GHRH gene, more preferably human GHRH. The 3xe2x80x2 flanking region is 3xe2x80x2 to the position for inserting coding sequence or the nucleic acid cassette.
The term xe2x80x9cflanking regionxe2x80x9d as used herein refers to nucleotide sequences on either side of an associated gene. Flanking regions can be either 3xe2x80x2 or 5xe2x80x2 to a particular gene in question. In general, flanking sequences contain elements necessary for regulation of expression of a particular gene. Such elements include, but are not limited to, sequences necessary for efficient expression, as well as tissue specific expression. Examples of sequences necessary for efficient expression can include specific regulatory sequences or elements adjacent to or within the protein coding regions of DNA. These elements, located adjacent to the gene, are termed cis-acting elements. The signals are recognized by other diffusible biomolecules in trans to alter the transcriptional activity. These biomolecules are termed trans-acting factors. Trans-acting factors and cis-acting elements have been shown to contribute to the timing and developmental expression pattern of a gene. Cis-acting elements are usually thought of as those that regulate transcription and are usually found within promoter regions and within upstream (5xe2x80x2) or downstream (3xe2x80x2) DNA flanking regions.
Flanking DNA with regulatory elements that regulate expression of genes in tissue may also include modulatory or regulatory sequences which are regulated by specific factors, such as glucocorticoids, androgens, progestins, antiprogestins (PCT US93/04399; PCT US96/04324), vitamin D3 and its metabolites, vitamin A and its metabolites, retinoic acid, calcium as well as others.
xe2x80x9cModulatoryxe2x80x9d or xe2x80x9cregulatoryxe2x80x9d sequences as used herein refer to sequences which may be in the 3xe2x80x2 or 5xe2x80x2 flanking region, where such sequences can enhance activation and/or suppression of the transcription of the associated gene.
xe2x80x9cResponsivexe2x80x9d or xe2x80x9crespondxe2x80x9d as used herein refers to the enhancement of activation and/or suppression of gene transcription as discussed below.
xe2x80x9cMetabolitexe2x80x9d as used herein refers to any product from the metabolism of the regulatory factors which Ace regulate gene expression.
In addition to the above, either or both of the 3xe2x80x2 or 5xe2x80x2 flanking regions can cause tissue specificity. Such tissue specificity provides expression predominantly in a specified cell or tissue.
xe2x80x9cPredominantlyxe2x80x9d as used herein means that the gene associated with the 3xe2x80x2 or 5xe2x80x2 flanking region is expressed to a higher degree only in the specific tissue as compared to low expression or lack of expression in nonspecific tissue. The 3xe2x80x2 or 5xe2x80x2 flanking regions singularly or together as used herein may provide expression of the associated gene in other tissues but to a lower degree than expression in tissues or cells where expression is predominate. Expression is preferentially in the specified tissue. Such predominant expression can be compared with the same magnitude of difference as will be found in the natural expression of the gene (i.e. as found in a cell in vivo) in that particular tissue or cell type as compared with other nonspecific tissues or cells. Such differences can be observed by analysis of mRNA levels or expression of natural gene products, recombinant gene products, or reporter genes. Furthermore, northern analysis, X gal immunofluorescence or CAT assays as discussed herein and as known in the art can be used to detect such differences.
The 3xe2x80x2 flanking region contains sequences or regions, e.g. 3xe2x80x2UTR and/or 3xe2x80x2 NCR, which regulate expression of a nucleic acid sequence with which it is associated. The 3xe2x80x2 flanking regions can provide tissue-specific expression to an associated gene. The 3xe2x80x2 flanking region also contains a transcriptional termination signal.
The term xe2x80x9c3xe2x80x2 flanking regionxe2x80x9d as used herein includes that portion of a naturally occurring sequence 3xe2x80x2 to the transcribed portion of the gene which are responsible for mRNA processing and/or tissue-specific expression. That portion can be readily defined by known procedures. For example, the portions of a 3xe2x80x2 flanking region which are responsible for mRNA stability and/or tissue-specific expression can be mapped by mutational analysis or various clones created to define the desired 3xe2x80x2 flanking region activity in a selected vector system.
The 3xe2x80x2 flanking region can contain a 3xe2x80x2UTR and/or a 3xe2x80x2 NCR. The term xe2x80x9c3xe2x80x2 untranslated regionxe2x80x9d or xe2x80x9c3xe2x80x2UTRxe2x80x9drefers to the sequence at the 3xe2x80x2 end of structural gene which is transcribed from the DNA but not translated into protein. This 3xe2x80x2UTR region does not contain a poly(A) sequence, but generally contains a site at which a poly(A) sequence is added. Poly (A) sequences are only added after the transcriptional process.
Myogenic-specific 3xe2x80x2UTR sequences can be used to allow for specific stability in muscle cells or other tissues. As described below, myogenic-specific sequences refers to gene sequences normally expressed in muscle cells, e.g., skeletal, heart and smooth muscle cells. Myogenic specific mRNA stability provides an increase in mRNA stability within myogenic cells. The increase in stability provides increased expression. The 3xe2x80x2UTR and 3xe2x80x2NCR sequences singularly or together can provide a higher level of mRNA accumulation through increased mRNA stability. Thus, increased expression and/or stability of mRNA leads to increased levels of protein production.
The term xe2x80x9c3xe2x80x2 non-coding regionxe2x80x9d or xe2x80x9c3xe2x80x2NCRxe2x80x9d is a region which is adjacent to the 3xe2x80x2UTR region of a structural gene. The 3xe2x80x2NCR region generally contains a transcription termination signal. Once transcription occurs and prior to translation, the RNA sequence encoded by the 3xe2x80x2NCR is usually removed so that the poly(A) sequence can be added to the mRNA. The 3xe2x80x2NCR sequences can also be used to allow mRNA stability as described above. The 3xe2x80x2NCR may also increase the transcription rate of the nucleic acid cassette.
Either or both of the 3xe2x80x2UTR and 3xe2x80x2NCR sequences can be selected from a group of myogenic-specific genes. Examples of myogenic-specific genes include the skeletal xcex1-actin gene, fast myosin-light chain 1/3 gene, myosin-heavy chain gene, troponin T gene, acetylcholine receptor subunit genes and muscle creatinine kinase gene.
In reference to 3xe2x80x2 flanking regions of this invention, the term xe2x80x9cgrowth hormonexe2x80x9d refers to a gene product identified as a growth hormone, for example, human growth hormone or bovine growth hormone. Homologous gene sequences are known in the art for a variety of different vertebrate animals. In different embodiments, the vectors can incorporate 3xe2x80x2 sequences, including 3xe2x80x2UTR sequences from such growth hormone genes. The 3xe2x80x2 sequence can be modified from the sequence naturally found in the animal, for example by the deletion of ALU repeat sequence from human growth hormone 3xe2x80x2UTR. The deletion of ALU repeats or ALU repeat-like sequences can be performed with a variety of 3xe2x80x2 sequences; such deletion generally reduces the rate of homologous recombination. A variety of other modifications may also be made without destroying the tissue targeting, stabilizing, and secretion properties of the 3xe2x80x2 sequence.
The 5xe2x80x2 flanking region is located 5xe2x80x2 to the associated gene or nucleic acid sequence to be expressed. As with the 3xe2x80x2 flanking region, the 5xe2x80x2 flanking region can be defined by known procedures. For example, the active portion of the 5xe2x80x2 flanking region can be mapped by mutational analysis or various clones of the 5xe2x80x2 flanking region created to define the desired activity in a selected vector. The 5xe2x80x2 flanking region can include, in addition to the above regulatory sequences or elements, a promoter, a TATA box, and a CAP site, which are in an appropriate relationship sequentially and positionally for the expression of an associated gene.
In this invention, xe2x80x9csequences necessary for expressionxe2x80x9d are those elements of the 5xe2x80x2 flanking region which are sequentially and positionally in an appropriate relationship to cause controlled expression of a gene within a nucleic acid cassette. Expression is controlled to certain levels within tissues such that the expressed gene is useful for gene therapy and other applications involving gene delivery. The 5xe2x80x2 sequence can contain elements which regulate tissue-specific expression or can include portions of a naturally occurring 5xe2x80x2 element responsible for tissue-specific expression.
The term xe2x80x9cpromoter,xe2x80x9d as used herein refers to a recognition site on a strand of DNA to which RNA polymerase binds. The promoter usually is a DNA fragment of about 100 to about 200 base pairs (in eukaryotic genes) in the 5xe2x80x2 flanking DNA upstream of the CAP site or the transcriptional initiation start site. The promoter forms an xe2x80x9cinitiation complexxe2x80x9d with RNA polymerase to initiate and drive transcriptional activity. The complex can be modified by activating sequences termed xe2x80x9cenhancersxe2x80x9d or inhibitory sequences termed xe2x80x9csilencersxe2x80x9d. The promoter can be one which is naturally (i.e., associated as if it were within a cell in vivo) or non-naturally associated with a 5xe2x80x2 flanking region.
A variety of promoters can be used. Some examples include thymidine kinase promoter, myogenic-specific promoters including skeletal xcex1-actin gene promoter, fast myosin light chain 1 promoter, myosin heavy chain promoter, troponin T promoter, and muscle creatinine kinase promoter, as well as non-specific promoters including the cytomegalovirus immediate early promoter, and Rous Sarcoma virus LTR. These promoters or other promoters used with the present invention can be mutated in order to increase expression of the associated gene. Furthermore a promoter may be used by itself or in combination with elements from other promoters, as well as various enhancers, transcript stabilizers, or other sequences capable of enhancing function of the vector.
xe2x80x9cMutationxe2x80x9d as used herein refers to a change in the sequence of genetic material from normal, causing a change in the functional characteristics of the gene. This includes gene mutations where only a single base is changed in the natural gene promoter sequences or multiple bases are changed.
The term xe2x80x9cintronxe2x80x9d as used herein refers to a section of DNA occurring in a transcribed portion of a gene which is included in a precursor RNA but is then excised during processing of the transcribed RNA before translation occurs. Intron sequences are therefore not found in mRNA nor translated into protein. The term xe2x80x9cexonxe2x80x9d as used herein refers to a portion of a gene that is included in a transcript of a gene and survives processing of the RNA in the cell to become part of a mature mRNA. Exons generally encode three distinct functional regions of the RNA transcript. The first, located at the 5xe2x80x2 end which is not translated into protein, termed the 5xe2x80x2 untranslated region (5xe2x80x2UTR), signals the beginning of RNA transcription and contains sequences that direct the mRNA to the ribosomes and cause the mRNA to be bound by ribosomes so that protein synthesis can occur. The second contains the information that can be translated into the amino acid sequence of the protein or function as a bioactive RNA molecule. The third, located at the 3xe2x80x2 end is not translated into protein, i.e. 3xe2x80x2UTR, contains the signals for termination of translation and for the addition of a polyadenylation tail (poly(A). In particular, the 3xe2x80x2UTR as defined above can provide mRNA stability. The intron/exon boundary will be that portion in a particular gene where an intron section connects to an exon section. The terms xe2x80x9cTATA boxxe2x80x9d and xe2x80x9cCAP sitexe2x80x9d are used as they are recognized in the art.
The term xe2x80x9clinkerxe2x80x9d as used herein refers to DNA which contains the recognition site for a specific restriction endonuclease. Linkers may be ligated to the ends of DNA fragments prepared by cleavage with some other enzyme. In particular, the linker provides a recognition site for inserting the nucleic acid cassette which contains a specific nucleic sequence to be expressed. This recognition site may be but is not limited to an endonuclease site in the linker, such as Cla-I, Not-I, Xmal, Bgl-II, Pac-I, Xhol, Nhel, Sfi-I. A linker can be designed so that the unique restriction endonuclease site contains a start codon (e.g. AUG) or stop codon (e.g. TAA, TGA, TCA) and these critical codons are reconstituted when a sequence encoding a protein is ligated into the linker. Such linkers commonly include an NcoI or SphI site.
The term xe2x80x9cleaderxe2x80x9d as used herein refers to a DNA sequence at the 5xe2x80x2 end of a structural gene which is transcribed and translated along with the gene. The leader usually results in the protein having an n-terminal peptide extension sometimes called a pro-sequence. For proteins destined for either secretion to the extracellular medium or the membrane, this signal sequence directs the protein into endoplasmic reticulum from which it is discharged to the appropriate destination. The leader sequence normally is encoded by the desired nucleic acid, synthetically derived or isolated from a different gene sequence. A variety of leader sequences from different proteins can be used in the vectors of the present invention. Some non-limiting examples include gelsolin, albumin, fibrinogen and other secreted serum proteins.
The term xe2x80x9cvectorxe2x80x9d as used herein refers to a nucleic acid, e.g., DNA derived from a plasmid, cosmid, phasmid or bacteriophage or synthesized by chemical or enzymatic means, into which one or more fragments of nucleic acid may be inserted or cloned which encode for particular genes. The vector can contain one or more unique restriction sites for this purpose, and may be capable of autonomous replication in a defined host or organism such that the cloned sequence is reproduced. The vector may have a linear, circular, or supercoiled configuration and may be complexed with other vectors or other materials for certain purposes. The components of a vector can include but are not limited to a DNA molecule incorporating: (1) a sequence encoding a therapeutic or desired product; and (2) regulatory elements for transcription, translation, RNA stability and replication.
The vector can be used to provide expression of a nucleic acid sequence in tissue. In the present invention this expression is enhanced by providing stability to an mRNA transcript from the nucleic acid sequence and/or secretion of the therapeutic protein. Expression includes the efficient transcription of an inserted gene or nucleic acid sequence within the vector. Expression products may be proteins including but not limited to pure protein (polypeptide), glycoprotein, lipoprotein, phosphoprotein, or nucleoprotein. Expression products may also be RNA. The nucleic acid sequence is contained in a nucleic acid cassette. Expression of the nucleic acid can be continuous or controlled by endogenous or exogenous stimuli.
The term xe2x80x9ccontrolxe2x80x9d or xe2x80x9ccontrolledxe2x80x9d as used herein relates to the expression of gene products (protein or RNA) at sufficiently high levels such that a therapeutic effect is obtained. Levels that are sufficient for therapeutic effect are lower than the toxic levels. Levels of expression for therapeutic effect within selected tissues corresponds to reproducible kinetics of uptake, elimination from cell, post-translational processing, and levels of gene expression, and, in certain instances, regulated expression in response to certain endogenous or exogenous stimuli (e.g., hormones, drugs).
The term xe2x80x9cnucleic acid cassettexe2x80x9d as used herein refers to the genetic material of interest which codes for a protein or RNA. The nucleic acid cassette is positionally and sequentially oriented within the vector such that the nucleic acid in the cassette can be transcribed into RNA, and when necessary, translated into a protein in the transformed tissue or cell. Preferably, the cassette has 3xe2x80x2 and 5xe2x80x2 ends adapted for ready insertion into a vector, e.g., it has restriction endonuclease sites at each end. In the vectors of this invention, a nucleic acid cassette contains a sequence coding for growth hormone releasing hormone (GHRH), e.g., human GHRH.
The term xe2x80x9ctissuexe2x80x9d as used herein refers to a collection of cells specialized to perform a particular function or can include a single cell. The cells may be of the same type or of different types.
The term xe2x80x9cgenexe2x80x9d, e.g., xe2x80x9cmyogenic genes,xe2x80x9d as used herein refers to those genes exemplified herein and their equivalence in other animal species or other tissues. Homologous sequences (i.e. sequences having a common evolutionary origin representing members of the same superfamily) or analogous sequences (i.e. sequences having common properties though a distinct evolutionary origin) are also included so long as they provide equivalent properties to those described herein. It is important in this invention that the chosen sequence provide the enhanced levels of expression, expression of the appropriate product, and/or tissue-specific expression as noted herein. Those in the art will recognize that the minimum sequences required for such functions are encompassed by the above definition. These minimum sequences are readily determined by standard techniques exemplified herein.
The term xe2x80x9cmyogenicxe2x80x9d refers to muscle tissue or cells. The muscle tissue or cells can be in vivo, in vitro, or in vitro tissue culture and capable of differentiating into muscle tissue. Myogenic cells include skeletal, heart and smooth muscle cells. Genes are termed xe2x80x9cmyogenicxe2x80x9d or xe2x80x9cmyogenic-specificxe2x80x9d if they are expressed or expressed preferentially in myogenic cells. Vectors are termed xe2x80x9cmyogenicxe2x80x9d or xe2x80x9cmyogenic-specificxe2x80x9d if they function preferentially in myogenic muscle tissue or cells. Myogenic activity of vectors can be determined by transfection of these vectors into myogenic cells in culture, injected into intact muscle tissue, or injected into mammalian oocytes to be stably incorporated into the genome to generate transgenic animals which express the protein or RNA of interest in myogenic cells.
The term xe2x80x9cnon-myogenicxe2x80x9d refers to tissue or cells other than muscle. The tissues or cells can be in vivo, in vitro, or in vitro tissue culture.
In a preferred embodiment, the vector described above may have its 5xe2x80x2 flanking region from myogenic genes, in particular the skeletal xcex1-actin gene, e.g., a chicken skeletal xcex1-actin gene. Specifically, this can include a promoter sequence which may be linked with other 5xe2x80x2UTR sequences, which can include an intron. While vectors using the chicken skeletal xcex1-actin promoter and/or other 5xe2x80x2 flanking sequences are exemplified herein, the 5xe2x80x2 sequences for xcex1-actin genes are highly conserved, therefore, such 5xe2x80x2 xcex1-actin sequences can be utilized from other vertebrate species, including, for example, human.
In preferred embodiments, the 3xe2x80x2UTR is from a growth hormone gene, preferably from a human growth hormone gene, and preferably includes a poly(A) signal. This sequence can be linked immediately following the natural translation termination codon for a cDNA sequence coding for the protein or RNA to be expressed. As discussed above, these regions can be further and more precisely defined by routine methodology, e.g., deletion or mutation analysis or their equivalents.
The 5xe2x80x2 or 3xe2x80x2 sequences may have a sequence identical to the sequence as naturally found, but may also have changed sequences which provide equivalent function to a vector in which such 5xe2x80x2 or 3xe2x80x2 sequences are incorporated. Such a change, for example, could be a change of ten nucleotides in any of the above regions. In particular, such changes can include the deletion of ALU repeat sequences from the 3xe2x80x2UTR. This is only an example and is non-limiting.
Also in preferred embodiments, the sequence encoding GHRH is a synthetic GHRH coding sequence. Such a synthetic sequence has a nucleotide sequence which differs from a natural human GHRH coding sequence. It is preferred that the sequence utilize optimal codon usage; preferably at least 50%, 70%, or 90% of the codons are optimized. Thus, in preferred embodiments the synthetic DNA sequence has at least 80, 90, 95, or 99% sequence identity to the sequence of SEQ ID NO. 1. In a particular preferred embodiment, the synthetic DNA sequence has at least 95% identity, more preferably at least 99% identity, and most preferably 100% identity to the sequence of SEQ ID NO. 2.
In addition, another embodiment of the above vector may contain a regulatory system for regulating expression of the nucleic acid cassette. The term xe2x80x9cregulatory systemxe2x80x9d as used herein refers to cis-acting or trans-acting sequences incorporated into the above vectors which regulate in some characteristic the expression of the nucleic acid of interest as well as trans-acting gene products which are co-expressed in the cell with the above described vector. Regulatory systems can be used for up-regulation or down regulation of expression from the normal levels of expression or existing levels of expression at the time of regulation. The system contributes to the timing and developmental expression pattern of the nucleic acid.
One construction of a regulatory system includes a chimeric trans-acting regulatory factor incorporating elements of a serum response factor capable of regulating expression of the vector in a cell. The chimeric transacting regulatory factor is constructed by replacing the normal DNA binding domain sequence of the serum response factor with a DNA binding domain sequence of a receptor. The serum response factor has a trans-activation domain which is unchanged. The trans-activation domain is capable of activating transcription when an agent or ligand specific to the receptor binding site binds to the receptor. Thus, regulation can be controlled by controlling the amount of the agent.
The DNA binding domain sequence of a receptor, incorporated into the chimeric trans-activating regulatory factor, can be selected from a variety of receptor groups including but not limited to vitamin, steroid, thyroid, orphan hormone, retinoic acid, thyroxine, or GAL4 receptors. The chimeric trans-activating regulator factor is usually located within the sequence of the promoter. In one preferred embodiment the promoter used in the vector is the xe2x96xa1-actin promoter and the receptor is the vitamin D receptor.
xe2x80x9cReceptorxe2x80x9d as used herein includes natural receptors (i.e., as found in a cell in vivo) as well as anything that binds alike and causes compartmentalization changes in a cell.
Another embodiment of the regulatory system includes the construction of a vector with two functional units. One functional unit expresses a receptor. This functional unit contains elements required for expression including a promoter, a nucleic acid sequence coding for the receptor, and a 3xe2x80x2UTR and/or a 3xe2x80x2NCR. The second functional unit expresses GHRH or a derivative or RNA and contains, in addition, a response element corresponding to the receptor, a promoter, a nucleic acid cassette, and a 3xe2x80x2UTR and/or a 3xe2x80x2NCR. These functional units can be in the same or separate vectors.
The first functional unit expresses the receptor. It is favorable to use a receptor not found in high levels in the target tissue. The receptor forms an interaction, e.g., ionic, non-ionic, hydrophobic, H-bonding, with the response element on the second functional unit prior to, concurrent with, or after the binding of the agent or ligand to the receptor. This interaction allows the regulation of the nucleic acid cassette expression. The receptor can be from the same nonlimiting group as disclosed above. Furthermore, the vector can be myogenic specific by using myogenic specific 3xe2x80x2UTR and/or 3xe2x80x2NCR sequences.
In an exemplary preferred embodiment the plasmid can be pSK-GHRH or a plasmid comprising a nucleotide sequence which is the same as the sequence of pSK-GHRH. This is only an example and is meant to be non-limiting. Thus, sequence changes or variations can be made to one or more of the sequence elements, such as the 5xe2x80x2 and 3xe2x80x2 flanking regions.
In this context, the word xe2x80x9csamexe2x80x9d means that the sequences are functionally equivalent and have a high degree of sequence identity. However, the sequences may have a low level of sequence differences, such as by substitution, deletion, or addition of one or more nucleotides. Such sequences will preferably be less than 10%, more preferably less than 5%, and still more preferably less than 1% of the total sequence.
In particular embodiments, the vectors of the above aspect may alternatively comprise, consist essentially of, or consist of the stated elements or sequences.
A related aspect of the invention provides a formulation for delivery and expression of a GHRH gene in a cell, preferably a human GHRH gene. The formulation includes a vector of the above aspect together with one or more other components which can, for example, act to stabilize the vector or to enhance transfection efficiency, but can also provide other functions. In a preferred embodiment, the formulation includes the vector in a solution having between about 0.5% and 50% polyvinyl pyrrolidone (PVP), preferably about 5% PVP. Preferably, the PVP has a weight average molecular weight of about 50,000 g/mol. Further information is disclosed in PCT US95/17038. However, another example of a formulation includes the vector with a cationic lipid (e.g., as described in U.S. Pat. No. 4,897,355, issued Jan. 30, 1990), and can also include a co-lipid, such as a neutral co-lipid, e.g., cholesterol.
In reference to the formulations of this invention, the term xe2x80x9caboutxe2x80x9d indicates that in preferred embodiments, the actual value for a particular parameter is in the range of 50%-200% of the stated value.
Another related aspect of the invention features a transgenic animal, at least some cells of which contain vectors of the first aspect of the present invention. These cells include germ or somatic cells. The transgenic animals can be used as models for studying human diseases, for assessing and exploring novel therapeutic methods, to assess the effects of chemical and physical carcinogens, and to study the effect of various genes and genetic regulatory elements. In addition, transgenic animals can be used to develop commercially important livestock species.
A fourth related aspect of the present invention features cells transformed with a vector of the present invention for expression of a GHRH nucleic acid sequence, preferably a human hGHRH (hGHRH) nucleic acid sequence.
As used herein, xe2x80x9ctransformationxe2x80x9d is the change in a cell""s phenotypic characteristics by the action of a gene expressing a gene product. The gene causing the phenotypic characteristic change has been transfected into the cell.
The term xe2x80x9ctransfectionxe2x80x9d as used herein refers to a mechanism of gene transfer which involves the uptake of DNA by a cell or organism. Following entry into the cell, the transforming DNA may recombine with that of the host by physically integrating into the chromosomes of the host cell, may be maintained transiently as an episomal element without being replicated, or may replicate independently as a plasmid. Preferably the transforming DNA does not integrate.
Transfection can be performed by in vivo techniques as described below, or by ex vivo techniques in which cells are co-transfected with a vector containing a selectable marker. This selectable marker is used to select those cells which have become transformed. It is well known to those skilled in the art the type of selectable markers to be used with transfection/transformation studies. An example of such a marker is a neo gene, providing neomycin/kanamycin resistance.
Transfection/transformation can be tissue-specific, i.e., involve the use of myogenic specific vectors which cause expression of the nucleic acid cassette predominantly in the tissue of choice. In particular, tissue specificity can be directed to myogenic cells by using a promoter and/or 3xe2x80x2UTR and/or 3xe2x80x2NCR sequences specific for myogenic tissue expression.
A fifth related aspect of the present invention features methods for transfecting a cell with the vectors of the present invention. These methods comprise the steps of contacting a cell in situ with a vector of the present invention for sufficient time to transfect the cell. As discussed above, transfection can be in vivo or ex vivo.
A sixth related aspect of the invention provides a method for delivery and expression of a GHRH gene, preferably a hGHRH gene. The method comprises transfecting a plurality of cells with a vector of the first aspect and incubating the cells under conditions allowing expression of a nucleic acid sequence of the vector, which codes for GHRH. The xe2x80x9cconditions allowing expressionxe2x80x9d may be any of a variety of conditions, including in vivo and in vitro conditions. Under such conditions, the cells will produce the gene product from the vector DNA in detectable quantities.
A seventh related aspect of the present invention features a method for treating a disease or condition by transfecting cells with the above-referenced vectors. Such disease or condition may, for example, be localized or systemic. These vectors contain nucleic acid sequences coding for growth hormone releasing hormone. Diseases and conditions can include but are not limited to burn, sepsis, trauma associated pathology, chronic obstructive pulmonary disease, aging associated osteoporosis, atherogenesis, atherosclerotic cardiovascular, cerebrovascular, or peripheral vascular disease, growth disorders and hemophilia.
The muscle atrophy to be treated may be due to any of a variety of different causes. For example, muscle weakness may be primarily due to disuse atrophy which commonly occurs in situations such as joint replacement, to muscle wasting during ageing, or to disease related cachexia. The causes may also include genetic causes of muscular atrophy, including, for example, muscular dystrophy. These causes and conditions are only exemplary and are not limiting to the invention.
Thus, xe2x80x9clocalizedxe2x80x9d disease or condition refers to those in which there is specific nerve or muscle damage or atrophy to a defined and limited area of the body. A specific example is disuse atrophy. A xe2x80x9csystemicxe2x80x9d disease or condition refers to those which relate to the entire organism, or is widely distributed at a number of locations within the body. Examples include growth disorders, neuropathies, and muscular dystrophy.
The methods of treating disease of the present invention feature methods for establishing expression of GHRH in tissue by administration of a vector. These methods of use of the above-referenced vectors comprise the steps of administering an effective amount of the vectors to a human, animal or tissue culture.
The term xe2x80x9cadministeringxe2x80x9d or xe2x80x9cadministrationxe2x80x9d as used herein refers to the route of introduction of a vector or carrier of DNA into the body. The vectors of the above methods and the methods discussed below may be administered by various routes. In particular a preferred target cell for treatment is the skeletal muscle cell.
The term xe2x80x9cskeletal musclexe2x80x9d as used herein refers to those cells which comprise the bulk of the body""s musculature, i.e., striated muscle.
Administration can be directly to a target tissue or may involve targeted delivery after systemic administration. The preferred embodiments are by direct injection into muscle or targeted uptake into muscle after intra-venous injection.
The term xe2x80x9cdeliveryxe2x80x9d refers to the process by which the vector comes into contact with the preferred target cell after administration. Administration may involve needle injection into cells, tissues, fluid spaces, or blood vessels, electroporation, transfection, hypospray, iontophoresis, particle bombardment, or transplantation of cells genetically modified ex vivo. Examples of administration include intravenous, intramuscular, aerosol, oral, topical, systemic, ocular, intraperitoneal and/or intrathecal.
The preferred means for administration of vectors described above involves the use of formulations for delivery to the target cell in which the vector is associated with elements such as lipids, proteins, carbohydrates, synthetic organic compounds, or inorganic compounds which enhance the entry of the vector into the nucleus of the target cell where gene expression may occur. A particular example is polyvinyl pyrrolidone(PVP).
The term xe2x80x9cformulationxe2x80x9d as used herein refers to non-genetic material combined with the vector in a solution, suspension, or colloid which enhances the delivery of the vector to a tissue, uptake by cells within the tissue, intracellular trafficking through the membrane, endosome or cytoplasm into the nucleus, the stability of the vector in extracellular or intracellular compartments, and/or expression of genetic material by the cell.
In a preferred embodiment of the present invention the vector and formulation comprises a nanoparticle which is administered as a suspension or colloid. The formulation can include lipids, proteins, carbohydrates, synthetic organic compounds, or inorganic compounds. Examples of elements which are included in a formulation are lipids capable of forming liposomes, cationic lipids, hydrophilic polymers, polycations (e.g. protamine, polybrine, spermidine, polylysine), peptide or synthetic ligand recognizing receptors on the surface of the target cells, peptide or synthetic ligand capable of inducing endosomal-lysis, peptide or synthetic ligand acapable of targeting materials to the nucleus, gels, slow release matrices, salts, carbohydrates, nutrients, or soluble or insoluble particles as well as analogues or derivatives of such elements. This includes formulation elements enhancing the delivery, uptake, stability, and/or expression of genetic material into cells. This list is included for illustration only and is not intended to be limiting in any way.
Another embodiment of the present invention features the above vectors with coating elements that enhance expression as well as uptake by the cell. The term xe2x80x9ccoatingxe2x80x9d as used herein refers to elements, proteins or molecules used to associate with the vector in order to enhance cellular uptake. In particular, coating includes a DNA initiation complex and histones. The coating improves the stability of the vector, its entry into the nucleus, and the efficiency of transcription.
The term xe2x80x9cDNA initiation complexxe2x80x9d as used herein refers to a complex containing a serum response factor, a transcription initiation factor and a trans-regulatory factor. The serum response factor is attached to or interacts with the serum response element within the promoter region of the vector. The transcription initiation factor and the trans-regulatory factor then interact with the serum response factor and the promoter, in particular the TATA box within the promoter, to form a stable DNA complex. The term xe2x80x9chistonexe2x80x9d as used herein refers to nuclear proteins which associate with and/or bind to DNA, e.g., a vector. The histones can bind specifically or non-specifically to the DNA.
The term xe2x80x9ceffective amountxe2x80x9d as used herein refers to sufficient vector administered to humans, animals or into tissue culture to produce the adequate levels of protein or RNA. One skilled in the art recognizes that the adequate level of protein or RNA will depend on the use of the particular vector. These levels will be different depending on the type of administration and treatment or vaccination.
The methods for treating diseases as disclosed herein includes treatment with biological products (specifically proteins as defined above) in which the disease being treated requires the protein to circulate through the body from the general circulation. For example, disorders which might be treated by the present invention include osteoporosis by expression of GHRH or its binding proteins. The selection of the appropriate protein to treat various diseases will be apparent to one skilled in the art.
In treating disease, the present invention provides a means for achieving: (1) sufficiently high levels of a particular protein to obtain a therapeutic effect; (2) controlled expression of product at levels which are sufficient for therapeutic effect and lower than the toxic levels; (3) controlled expression in certain tissues in order to obtain reproducible pharmacokinetics and levels of gene expression; and (4) delivery using clinically and pharmaceutically acceptable means of administration and formulation rather than transplantation of genetically engineered and selected cells.
In doing so, the present invention provides significant advances over the art. First, promoters from viral genomes and viral vectors which were used to obtain high level expression in tissue, were not able to provide controlled expression. Second, promoters from various tissue-specific genes which were used to obtain controlled expression in transgenic animals and animal models of gene therapy did not have a sufficiently high level of expression to obtain therapeutic effect. In addition, in treating diseases with the present invention, the ability to raise antibodies against protein products does not reflect the ability to achieve controlled expression of proteins within the therapeutic range.
An eighth related aspect of the present invention features a method of gene replacement for inherited genetic diseases of muscle. This method includes the transfection of muscle cells with the above-referenced vectors.
The genetic material which is incorporated into the cells from the above vectors can be any natural or synthetic nucleic acid. For example, the nucleic acid can be: (1) not normally found in the tissue of the cells; (2) normally found in a specific tissue but not expressed at physiological significant levels; (3) normally found in specific tissue and normally expressed at physiological desired levels; (4) any other nucleic acid which can be modified for expression in skeletal muscle cells; and (5) any combination of the above. In addition to the genetic material which is incorporated into tissue, the above reference is also applicable to genetic material which is incorporated into a cell.
By xe2x80x9ccomprisingxe2x80x9d it is meant including, but not limited to, whatever follows the word xe2x80x9ccomprisingxe2x80x9d. Thus, use of the term xe2x80x9ccomprisingxe2x80x9d indicates that the listed elements are required or mandatory, but that other elements are optional and may or may not be present. By xe2x80x9cconsisting ofxe2x80x9d is meant including, and limited to, whatever follows the phrase xe2x80x9cconsisting ofxe2x80x9d. Thus, the phrase xe2x80x9cconsisting ofxe2x80x9d indicates that the listed elements are required or mandatory, and that no other elements may be present. By xe2x80x9cconsisting essentially ofxe2x80x9d is meant including any elements listed after the phrase, and limited to other elements that do not interfere with or contribute to the activity or action specified in the disclosure for the listed elements. Thus, the phrase xe2x80x9cconsisting essentially ofxe2x80x9d indicates that the listed elements are required or mandatory, but that other elements are optional and may or may not be present depending upon whether or not they affect the activity or action of the listed elements.
The present invention also concerns a gene therapy approach in which a species-specific GHRH cDNA plasmid based expression vector or other species-specific GHRH expression vectors are targeted into peripheral organs and expressed by the transfected cells. The species-specific GHRH polypeptide is then processed, secreted and transported to the anterior pituitary, where it stimulates GH release.
As used herein, a xe2x80x9cplasmidxe2x80x9d is an extrachromosomal genetic element consisting of a circular duplex of DNA which can replicate independently of chromosomal DNA. Plasmids are used in gene transfer, as the vehicle by means of which DNA fragments can be introduced into a host organism, and are associated with the transfer of antibiotic resistance.
The present invention concerns a method of expressing growth hormone releasing hormone (GHRH) in a non-human vertebrate animal comprising the step of inserting a DNA carrier vehicle containing a gene sequence encoding a growth hormone releasing hormone polypeptide sequence operatively linked to a vertebrate gene promoter into said non-human vertebrate animal tissue under conditions where said gene (a segment of DNA which codes for a specific polypeptide or RNA molecule) is expressed and produces hormone releasing hormone.
The term xe2x80x9cnon-human vertebrate animalxe2x80x9d encompasses all animals having a backbone or spinal column, except for human beings. Vertebrate animals include fishes, amphibians, reptiles, birds and mammals.
As used herein, a xe2x80x9cDNA carrier vehiclexe2x80x9d refers to some means by which DNA fragments can be introduced into a host organism or host tissue. The DNA carrier vehicle may be designed to incorporate the gene of interest and any accessory genetic sequences.
The xe2x80x9cgene sequencexe2x80x9d preferably is a nucleic acid molecule.
The present invention concerns gene sequences that encode for a growth hormone releasing hormone having one of the following sequences; SEQ ID NO.5, SEQ ID NO.6, SEQ ID NO.7, SEQ ID NO.8, SEQ ID NO.9, or SEQ ID NO.11; or where the non-human vertebrate animal is one of the following species; porcine, bovine, equine, canine, feline, caprine, avian (chicken, turkey, duck), ovine or fish.
The present invention also can involve GHRH gene sequence in the DNA carrier vehicle that contain no intervening sequences in the GHRH region.
A further object of the invention is use of a DNA carrier vehicle in which the promoter is from a skeletal xcex1-actin gene.
The present invention includes a DNA carrier vehicle which is injected into said animal muscle.
The present invention includes a plasmid DNA vector, adenovirus or adeno-associated virus as DNA carrier vehicles capable of infecting non-human vertebrate animals in various tissues.
The present invention can include a DNA carrier vehicle in which the promoter-GHRHcDNA-3xe2x80x2UTR is incorporated into an adeno-associated virus. The present invention can additionally include an embodiment where the vectors encode for an Argxe2x80x94Arg sequence before a tyrosine or a histidine.
A further object of the invention is incorporation of a gene switch sequence into the DNA carrier vehicle.
The present invention also includes a gene sequence which is a chimeric synthetic cDNA encoding GHRH comprising a mouse specific fragment and a species specific fragment and wherein the mouse specific fragment contains the first 45 nucleotides and encodes the first 15 amino acids of the mouse GHRH, and said mouse specific fragment is fused in frame with a species-specific fragment containing 87 nucleotides which encode the 16th to 44th amino acids of a species-specific GHRH. This chimeric sequence provides resistance against dipeptidases.
As used herein, a xe2x80x9cchimeraxe2x80x9d refers to a molecule with genetic material from genetically different organisms.
Furthermore, the present invention can include a species-specific fragment for GHRH encoding a polypeptide from one of the following animal species; porcine, bovine, equine, canine, feline, caprine, avian (chicken, turkey, duck), ovine or fish or encoding for one of the following GHRH polypeptides; SEQ ID NO.5, SEQ ID NO.6, SEQ ID NO.7, SEQ ID NO.8, SEQ ID NO.9, or SEQ 2 Ed ID NO.11.
The present invention also concerns a method whereby skeletal muscle can be transfected in vivo by direct plasmid DNA injection or direct injection of other DNA carrier vehicles.
The present invention further concerns growth promoting myogenic expression plasmid vectors, pSK-GHRH, that drive high-level GHRH expression from animal muscle.
For example, species-specific GHRH can be secreted in vitro, in primary chicken and pig myotube cultures, and in vivo, after the intramuscular injection into regenerating quadriceps muscle of immunocompetent adult C57/B16 mice, or in vivo in the appropriate vertebrate species. Intramuscular injection of pSK-GHRH results in increased serum mGH, several fold over control values, for at least two weeks, and increased liver IGF-1 mRNA levels and enhances animal growth as compared to control animals.
The invention features a plasmid DNA based system which contains a vertebrate gene promoter that provides constitutive transcriptional activity. Other suitable vectors may also be used. The invention may also utilize muscle or other tissue specific promoters, or viral promoters active in animal cells. Human GHRH sequence is not desirable for use in other vertebrate species because it is antigenic and produces antibodies following injection into lower vertebrate animals. The DNA of the invention contains no intervening sequences anywhere in the plasmid DNA. The invention will use the target tissue""s transcription, translation and secretory activities to transcribe the GHRH mRNA, correctly translate and then process the GHRH precursor protein, which, in turn, allows for secretion into the systemic blood supply. The increased levels of secreted GHRH will stimulate secretion of GH from the target animal""s anterior pituitary.
Several embodiments of the invention involve GHRH expression for ectopic expression of a truncated GHRH from muscle, liver, heart, lung and vascular tissues by a plasmid DNA vector. This vector may contain eukaryotic promoters including various cell or tissue specific promoters (e.g., muscle, endothelial cell, liver), various viral promoters and enhancers, and various GHRH cDNAs isogenically specific for each animal species including porcine, equine, bovine, canine, feline, caprine, ovine, avian (chicken, turkey, duck) or fish. The vector may also contain a chimeric GHRH cDNA composed of the first 15 amino acids of the mouse GHRH following the processed N-terminal histidine, numbered 1, fused in frame with a specific animal GHRH species fragment covering amino acid 16 up to 44 amino acids, a synthetic stop codon, an SV40 or a growth hormone 3xe2x80x2 untranslated region containing polyadenylation sites. Any and all of these embodiments may utilize suitable vectors other than a plasmid DNA.
The plasmid of the invention may be incorporated into adenoviral and adenoviral-associated viruses and injected into muscle or into the blood system. DNA taken up into the cellular nuclei of tissue allows for transcription of a messenger RNA encoding a truncated GHRH polypeptide which is then translated into a precursor GHRH protein. The precursor protein requires metalloprotease or other processing to allow for a biologically active GHRH to be secreted. The precursor protein is trimmed to either a N-terminal tyrosine or a N-terminal histidine depending upon the animal GHRH species. Ectopic secretion from muscle and other tissues including liver, pancreas, kidney and heart of the correctly processed GHRH into the blood system, increases the concentration of GHRH in the blood, which then causes a profound stimulation of growth hormone (GH) secretion from the anterior pituitary of the target animal. Skeletal muscle-secreted GHRH is biologically active, as demonstrated by eliciting robust GH release following a single intramuscular injection of 100xcexc plasmid CMV-GHRH DNA sufficient to elevate GH levels 3 to 4 fold for up to 2 weeks, to enhance liver IFG-1 gene expression and to increase body weight approximately 10%. Thus, plasmid based-GHRH can-serve as a potent GH secretagogue in animals.
One embodiment of the invention includes a novel plasmid vector which is capable of directing high-level gene expression in a skeletal muscle specific manner. A 228 bp fragment of hGHRH, which encodes for the 31 amino acid signal peptide and the entire mature peptide hGHRH(1-44)OH(Tyr1xe2x86x92Leu44) is cloned into a pBS-derived vector. Gene expression is controlled by a 448 bp fragment (xe2x88x92424/+24) of the avian skeletal xcex1-actin gene, which contains several evolutionarily conserved regulatory elements that accurately initiate skeletal xcex1-actin transcripts and drives transcription of a variety of reporter genes specifically in differentiated skeletal muscle cells. The GHRH coding region is followed by the 3xe2x80x2 untranslated region of human growth hormone cDNA.
In another embodiment the cytomegalovirus promoter and enhancer is used. In a preferred embodiment the promoter is linked to a synthetic GHRH cDNA which contains any non-muscle 3xe2x80x2 untranslated region cDNA. In one preferred embodiment a bovine growth hormone 3xe2x80x2 untranslated region cDNA is used.
In one embodiment of the invention the plasmid DNA is injected into muscle. In yet another preferred embodiment the promoter GHRHcDNA-3xe2x80x2UTR can be incorporated into a virus, such as an adeno-associated virus for viral infection of muscle.
In another embodiment the invention is incorporated in adenoviruses and allowed to infect a variety of tissues that will then express species specific GHRH mRNA in any tissue the adenovirus infects.
In another embodiment the GHRH vectors are made with species specific GHRH that contains a metalloenzyme processing sequence Arginine-Arginine before a Tyrosine or a Histidine. Each GHRH polypeptide secreted can be made isogenic so that it is identical to the actual animals"" GHRH.
Another embodiment of the invention employs a gene switch element in the DNA carrier vehicle. switch element in the DNA carrier vehicle.
In another embodiment a chimera synthetic cDNA encoding GHRH is used that contains the first 45 nucleotides to encode the first 15 amino acids of the mouse GHRH fused in frame with the nucleic acid sequence encoding the 16th to the 44th amino acids of the species-specific GHRH cDNA sequence. This chimeric sequence is effective in providing resistance against proteolytic degradation by dipeptidases.
In other embodiments of the invention the animal species of GHRH encoded for may include: porcine, bovine, equine, canine, feline, caprine, avian (chicken, turkey, duck), ovine and fish.
Other embodiments of the invention utilize nucleotides sequences coding for one of the GHRH polypeptides expressed by any one of SEQ ID NO.5, SEQ ID NO.6, SEQ ID NO.7, SEQ ID NO.8, SEQ ID NO.9, and SEQ ID NO.11. A skilled artisan will readily recognize that these polypeptides can be encoded for by a number of nucleotide sequences.
Other features and advantages of the invention will be apparent from the following detailed description of the invention in conjunction with the accompanying drawings and from the claims.